Evaluation of an affordable HIV-1 virological failure assay and antiretroviral drug resistance genotyping protocol

•A virological failure (VF) and HIV drug resistance (HIVDR) assay was evaluated.•248 subtype C samples from patients failing first line therapy were tested.•VF assay performed well on both plasma and DBS, measured against a reference assay.•HIVDR assay showed reliable amplification and sequencing results.•These assays are simple and viable options to detect virological failures. HIV-1 RNA viral load is the preferred tool to monitor virological failure during antiretroviral therapy (ART) exposure. Timely detection of virological failure can reduce the prevalence and complexity of HIV-1 drug resistance. This field evaluation further characterizes a two-step approach to identify virological failure, as a measure of ART adherence, and detect HIVDR mutations in the reverse transcriptase (RT) gene of HIV-1. Two hundred and forty-eight (248) samples were tested; 225 from South African HIV-1 participants enrolled in the PharmAccess African Studies to Evaluate Resistance (PASER) cohort, forty of which had paired dried blood spot (DBS) samples and 23 HIV-1 negative samples. A newly developed virological failure assay (ARTA-VFA) was used on all samples, and those with a viral load >5000 RNA copies/ml were genotyped with a shortened RT protocol to detect HIVDR (ARTA-HIVDRultralight). The ARTA-VFA showed good precision and linearity as compared to a commercial reference assay (NucliSENS® EasyQ v1.2, Roche) with an R2 of 0.99. Accuracy studies illustrated standard deviations of